Typing in Situ with Probes

Abstract

After nearly one hundred years of fruitless searches for methods for the establishment of prokaryotic systematics based on genealogical relationships of the organisms, Zuckerkandl and Pauling (1965) introduced the idea of “Molecules as documents of evolutionary history”. Thereafter the “molecular revolution” (Woese, 1991) was launched by Woese and coworkers who introduced comparative sequence analysis of ribosomal RNA for the elucidation of the phylogeny of microorganisms (Fox et al., 1977; Woese and Fox, 1977). Nowadays, the determination of rRNA primary structures has become a routine method for taxonomic investigations. The number of small subunit rRNA sequences which are currently available in public databases (Larsen et al., 1993; Neefs et al., 1993) is about 2000, that of large subunit counterparts about 200; both are rapidly growing. The larger data set allowed not only definition of groups of phylogenetically related microorganisms but also recognition of molecular signatures (Woese, 1987) for these groups. Signatures are rRNA structure elements which are unique to a particular group. Primary structure elements, such as single nucleotides or sequence stretches, insertions (Roller et al., 1992) and deletions (Ludwig et al., in press), as well as presence and shape of higher order structure elements (Roller et al., 1992; Ludwig et al., 1992) may represent signatures for higher or lower phylogenetic entities respectively. Primary structure signatures can be used as target sites for diagnostic hybridization probes, thereby allowing identification of microorganisms on the basis of their phylogenetic relationships. Since the first experimental approaches (Göbel and Stanbridge, 1984; Festl et al., 1986), a reasonable number of rRNA or rDNA (DNA encoding rRNA) targeted, specific hybridization probes has been developed (reviewed by Schleifer et al., 1993).